Method for producing galactosylceramide and filamentous fungus

Abstract

To provide a method for producing galactosylceramide using enzyme derived from filamentous fungus.SOLUTION: A method for producing galactosylceramide includes a step of contacting glucosylceramide with any of the following enzymes (a) to (c) to convert glucosylceramide to galactosylceramide. (a) An enzyme containing a specific amino acid sequence. (b) An enzyme containing an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in a specific amino acid sequence, and having the activity of converting glucosylceramide to galactosylceramide. (c) An enzyme containing an amino acid sequence having 60% or more sequence identity with a specific amino acid sequence and having the activity of converting glucosylceramide to galactosylceramide.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing galactosylceramide and a filamentous fungus. [Background technology]

[0002] Glucosylceramide and galactosylceramide are known as sphingoglycolipids that constitute the cell membranes of human cells. Glucosylceramide has a moisturizing effect on the skin and is used in cosmetics and the like.

[0003] On the other hand, it is known that glucosylceramide also exists as a membrane component in filamentous fungi, and Non-Patent Document 1 describes that Aspergillus oryzae, a type of filamentous fungus, has galactosylceramide. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Tani Y., et al., Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae. Biotechnol Letters, 36, 2507-2513, 2014. Summary of the Invention [Problem to be solved by the invention]

[0005] However, the enzymes involved in the biosynthesis of galactosylceramide in filamentous fungi have not been elucidated, and no technology for producing galactosylceramide using filamentous fungi has existed until now.

[0006] An object of the present invention is to provide a method for producing galactosylceramide using an enzyme derived from a filamentous fungus and a filamentous fungus capable of mass-producing galactosylceramide. [Means for solving the problem]

[0007] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0008] [1] A method for producing galactosylceramide, comprising the step of contacting glucosylceramide with any one of the following enzymes (a) to (c) to convert glucosylceramide to galactosylceramide: (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11; (b) an enzyme comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 11, and having activity of converting glucosylceramide to the galactosylceramide; (c) an enzyme comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 11, and having activity of converting glucosylceramide to the galactosylceramide. [2] The method for producing galactosylceramide described in [1], wherein the step of converting glucosylceramide to galactosylceramide is carried out within the cells of a filamentous fungus expressing any one of the enzymes (a) to (c).

[0009] [3] A method for producing galactosylceramide according to [2], comprising the step of irradiating the filamentous fungus with ultraviolet light.

[0010] [4] A method for producing galactosylceramide described in [2], which includes a step of transforming the filamentous fungus so that it overexpresses any one of the enzymes (a) to (c) prior to the step of converting glucosylceramide to galactosylceramide.

[0011] [5] A filamentous fungus transformed to overexpress any one of the following enzymes (a) to (c): (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11; (b) an enzyme comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 11, and having activity of converting glucosylceramide to galactosylceramide; (c) an enzyme comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 11, and having activity of converting glucosylceramide to galactosylceramide. Effect of the Invention

[0012] According to the present invention, there are provided a method for producing galactosylceramide using an enzyme derived from a filamentous fungus, and a filamentous fungus capable of mass-producing galactosylceramide. [Brief description of the drawings]

[0013] [Figure 1] 1 is a flow chart showing a method for producing galactosylceramide according to a preferred embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic showing the sequence of the first plasmid used in the transformation process. [Diagram 3] FIG. 2 is a schematic showing the sequence of the second plasmid used in the transformation process. [Figure 4] Chromatographs of glycolipids obtained from various strains of Aspergillus oryzae. [Diagram 5] 1 is a chromatogram showing the result of reacting endogalactosylceramidase with glycolipids obtained from Aspergillus oryzae. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings where necessary.

[0015] <<How to produce galactosylceramide>> In one embodiment, the present invention provides a method for producing galactosylceramide, comprising the step of contacting glucosylceramide with any one of the following enzymes (a) to (c) to convert the glucosylceramide into galactosylceramide: (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11 (b) an enzyme comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to the galactosylceramide; (c) an enzyme comprising an amino acid sequence having 60% or more sequence identity with any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to galactosylceramide;

[0016] FIG. 1 is a flow diagram showing a method for producing galactosylceramide according to a preferred embodiment of the present invention.

[0017] As shown in Fig. 1, the production method of this embodiment includes a step of transforming a filamentous fungus (transformation step S1), a step of culturing the transformed filamentous fungus and synthesizing galactosylceramide (synthesis step S2), and a step of extracting the synthesized galactosylceramide from the filamentous fungus (extraction step S3). Note that the production method of galactosylceramide does not necessarily need to include the above-mentioned transformation step S1 and extraction step S3, and it is sufficient if it includes at least the synthesis step S2.

[0018] [Transformation step S1] The transformation step S1 is a step of transforming a filamentous fungus so as to overexpress any one of the following enzymes (a) to (c): (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11 (b) an enzyme comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to galactosylceramide; (c) an enzyme comprising an amino acid sequence having 60% or more sequence identity with any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to galactosylceramide;

[0019] The present inventors have found that filamentous fungi do not contain any enzymes that are homologous to galactosyltransferases that add galactose to ceramide and that are known in higher animals.

[0020] After that, the present inventors, through trial and error, discovered that in filamentous fungi, the enzymes containing the amino acid sequences shown in SEQ ID NOs: 1 to 11 have the activity of converting glucosylceramide to galactosylceramide. Hereinafter, the enzymes (a) to (c) are referred to as "galactosylceramide synthase" or "GceA", respectively.

[0021] In the transformation step S1, a gene encoding any one of the galactosylceramide synthases (a) to (c) is introduced into a filamentous fungus to overexpress the galactosylceramide synthase compared to a wild-type strain, thereby enabling mass production of galactosylceramide.

[0022] <Galactosylceramide synthase (a)> The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of galactosylceramide synthase of the RIB40 strain of Aspergillus oryzae. This galactosylceramide synthase is encoded in the RIB40 strain of Aspergillus oryzae by the DNA sequence shown in SEQ ID NO: 12 (Genbank: NC_036442.1). The gene is present on chromosome 8 of Aspergillus oryzae.

[0023] The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of galactosylceramide synthase in Aspergillus fumigatus. In Aspergillus fumigatus, this galactosylceramide synthase is encoded by the base sequence shown in SEQ ID NO: 13. The gene is present on chromosome 2 of Aspergillus fumigatus.

[0024] The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of galactosylceramide synthase in Aspergillus niger. This galactosylceramide synthase is encoded in Aspergillus niger by the DNA sequence shown in SEQ ID NO: 14.

[0025] The amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of galactosylceramide synthase in Aspergillus nidulans. In Aspergillus nidulans, this galactosylceramide synthase is encoded by the DNA sequence shown in SEQ ID NO: 15. The gene is present on chromosome 7 of Aspergillus nidulans.

[0026] The amino acid sequence shown in SEQ ID NO:5 is the amino acid sequence of galactosylceramide synthase in Penicillium camemberti (the bacterium that produces Camembert cheese).

[0027] The amino acid sequence shown in SEQ ID NO:6 is the amino acid sequence of galactosylceramide synthase in Aureobasidium pullulans (a bacterium that produces the polysaccharide pullulan).

[0028] The amino acid sequence shown in SEQ ID NO: 7 is the amino acid sequence of galactosylceramide synthase in Cordyceps militaris (a type of cordyceps mushroom).

[0029] The amino acid sequence shown in SEQ ID NO:8 is the amino acid sequence of galactosylceramide synthase in Beauveria bassiana (an insect parasitic fungus).

[0030] The amino acid sequence shown in SEQ ID NO: 9 is the amino acid sequence of galactosylceramide synthase in Fusarium oxysporum (a plant pathogen).

[0031] The amino acid sequence shown in SEQ ID NO: 10 is the amino acid sequence of galactosylceramide synthase in Trichoderma atroviride (a plant pathogen).

[0032] The amino acid sequence shown in SEQ ID NO: 11 is the amino acid sequence of galactosylceramide synthase in Glonium stellatum (Modulus stellatum).

[0033] These galactosylceramide synthases convert glucosylceramide to galactosylceramide by epimerizing the hydroxyl group attached to the 4-carbon of the glycosyl group of glucosylceramide.

[0034] <Galactosylceramide synthase (b)> With regard to (b) above, "an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which one or more and 10 or less, preferably one or more and 8 or less, more preferably one or more and 5 or less, and even more preferably one or more and 3 or less amino acids have been deleted, substituted, added, or inserted.

[0035] <Galactosylceramide synthase (c)> In the present specification, the sequence identity of a subject amino acid sequence to a reference amino acid sequence (the amino acid sequence shown in SEQ ID NOs: 1 to 11 above) can be determined, for example, as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Each amino acid sequence may contain gaps so as to maximize the sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (1).

[0036] Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100… (1) The galactosylceramide synthase (c) comprises an amino acid sequence having 60% or more sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 1 to 11, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence identity.

[0037] <Gene introduction> The method for introducing a gene encoding any one of the galactosylceramide synthases (a) to (c) into a filamentous fungus is not particularly limited, but may be, for example, by incorporating the gene into a plasmid DNA.

[0038] When plasmid DNA is used, high expression of galactosylceramide synthase in filamentous fungi can be achieved by incorporating a constitutive promoter or an inducible promoter in combination.

[0039] Examples of constitutive promoters include Ptef, which is a promoter of the tef1 gene, PpgkA, which is a promoter of the pgkA gene, and the enoA promoter, etc. Ptef is a promoter for strong expression, and PpgkA is a promoter for moderately high expression.

[0040] Examples of inducible promoters include the amyB promoter, which uses maltose as an induction substrate, and the xylanase promoter, which uses xylose as an induction substrate.

[0041] Methods for introducing plasmid DNA into filamentous fungi include, but are not limited to, electroporation and protoplast PEG methods.

[0042] The organism into which the plasmid DNA is introduced is not particularly limited as long as it is a filamentous fungus.

[0043] The galactosylceramide synthase gene to be incorporated into the plasmid DNA may be a gene encoding a galactosylceramide synthase of the same species as the target to be introduced, or it may be a gene encoding a galactosylceramide synthase of another type of filamentous fungus (any of the enzymes (a) to (c) above).

[0044] [Synthesis step S2] In the synthesis step S2, the filamentous fungus transformed in the transformation step S1 is cultured to synthesize galactosylceramide.

[0045] In the synthesis step S2, the transformed filamentous fungus is cultured, and the overexpressed galactosylceramide synthase naturally comes into contact with glucosylceramide in the fungal body and converts glucosylceramide to galactosylceramide. Galactosylceramide is considered to be a sphingoglycolipid that penetrates the membrane three times, and the synthesized galactosylceramide is located in the cell membrane.

[0046] The type of medium for culturing filamentous fungi is appropriately selected depending on the fungal species. When culturing Aspergillus oryzae, for example, CD medium or DPY liquid medium used in the Examples below can be used.

[0047] In addition, if an inducible promoter is introduced into the filamentous fungus together with galactosylceramide synthase in the transformation step S1, in order to overexpress galactosylceramide synthase, it is necessary to add an inducing substrate such as maltose or xylose to the medium in which the filamentous fungus is cultured in the synthesis step S2.

[0048] [Extraction process S3] The method for extracting galactosylceramide synthesized by filamentous fungi is not particularly limited, and for example, a known extraction method using an organic solvent can be used.

[0049] For example, JP 2023-064103 A discloses a method for extracting ceramide by collecting fungal cells from a culture solution of a filamentous fungus, drying the fungal cells, adding an aqueous ethanol solution and stirring to extract the mixture, distilling the resulting extract under reduced pressure at 45°C to obtain a concentrated extract, and then washing with acetone.

[0050] Furthermore, JP 2012-126910 A discloses a method for extracting ceramide powder by adding ethanol to soy sauce cake containing ceramide, stirring and extracting the mixture, concentrating and drying the mixture in a rotary evaporator, washing with acetone, re-extracting the mixture with ethanol, removing impurities using activated carbon or anion exchange resin, dissolving the resulting re-extract in ethanol, adding water, precipitating the mixture by cooling and centrifuging, washing the mixture with acetone or the like, and drying the mixture in a vacuum desiccator.

[0051] Furthermore, the extraction step S3 may include a step of recovering conidia of a filamentous fungus, a step of inoculating and culturing the recovered conidia, and a step of extracting glycolipids from the fungal cells cultured from the conidia, as described in detail in the Examples.

[0052] <<Filamentous fungi>> In one embodiment, the present invention provides a filamentous fungus transformed to overexpress any one of the following enzymes (a) to (c), where the following enzymes (a) to (c) are the same as those described above: (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11 (b) an enzyme comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to galactosylceramide; (c) an enzyme comprising an amino acid sequence having 60% or more sequence identity with any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to galactosylceramide;

[0053] The sequence identity in (c) above is preferably 80% or more, more preferably 90% or more, and most preferably 95% or more.

[0054] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included in the scope of the present invention.

[0055] For example, in the embodiment shown in Figure 1, galactosylceramide was synthesized within the cells of a filamentous fungus in synthesis step S2, but any of the enzymes (a) to (c) above may be isolated from a filamentous fungus and contacted with glucosylceramide in vitro to convert it to galactosylceramide.

[0056] 1 includes a transformation step S1, but may include a step of irradiating the filamentous fungus with ultraviolet light instead of or in addition to the transformation step S1. The step of irradiating the filamentous fungus with ultraviolet light is carried out prior to the synthesis step S2.

[0057] The present inventors have discovered that the expression of galactosylceramide synthase can be increased by irradiating the RIB40 strain of Aspergillus oryzae, a type of filamentous fungus, with ultraviolet light, and have found the possibility of mass-producing galactosylceramide from glucosylceramide even in wild-type strains. The RIB40 strain has a gene encoding the galactosylceramide synthase having the amino acid sequence shown in SEQ ID NO: 1, but the gene is not normally expressed. EXAMPLES

[0058] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0059] [Transformation process] FIG. 2 is a schematic diagram showing the sequence of the first plasmid used in the transformation process, and FIG. 3 is a schematic diagram showing the sequence of the second plasmid used in the transformation process.

[0060] In this example, first, the plasmid having the base sequence shown in SEQ ID NO: 16 (see FIG. 2) or the plasmid having the base sequence shown in SEQ ID NO: 17 (see FIG. 3) was introduced into the RIB40 strain of Aspergillus oryzae for transformation.

[0061] Aspergillus oryzae has an ATP sulfurylase gene that has been previously mutated, allowing it to grow only in a medium containing methionine. The parent strain of Aspergillus oryzae used in the transformation of this example is NS1DN1(niaD + ,sC - ) was.

[0062] The first plasmid shown in Figure 2 contains the above-mentioned Ptef, the gceA gene encoding galactosylceramide synthase (see sequence number 12), TamyB, which is the terminator of amyB, and a partial sequence (sC) of AosC, which is a selection marker.

[0063] Aspergillus oryzae into which this plasmid has been introduced has a sequence that allows AosC to function normally through homologous recombination, enabling the plant to survive in a medium that does not contain methionine.

[0064] The second plasmid shown in FIG. 3 contains the above-mentioned PpgkA in place of the Ptef of the first plasmid.

[0065] In addition to Aspergillus oryzae strains into which either of the above two types of plasmids had been introduced, we also prepared a wild-type strain (WT) of Aspergillus oryzae RIB40 and a control (Ctr) strain into which a plasmid containing the selection marker sC but not the gceA gene had been introduced.

[0066] The genotype of each line is shown in Table 1 below.

[0067] [Table 1]

[0068] In Table 1, NSlDN1(host) is the target host strain (before introduction) into which the above-mentioned two types of plasmids containing the gceA gene were introduced.

[0069] [Synthesis process] Each of the above strains of Aspergillus oryzae was subcultured on CD medium.

[0070] CD medium contained 0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, and 2% glucose, and had a pH of 5.5.

[0071] Aspergillus oryzae was cultured on the plate containing the medium for 4 days.

[0072] [Extraction process] 10 mL of sterilized 0.01% Tween 20 (Merck) was added to the plate on which conidia had formed, and the conidia were suspended using a sterilized spreader.

[0073] The resulting suspension was filtered using Miracloth (Merck) to remove cell debris, which was then collected in a 15 mL tube and centrifuged at 2000 rpm for 5 minutes.

[0074] After centrifugation, the supernatant was discarded, 5 mL of sterile water was added, and the conidia were suspended by vibration stirring, followed by centrifugation again at 2000 rpm for 5 minutes.

[0075] The supernatant was then discarded, and sterile water was added to suspend the conidia, which were then transferred to a 2 mL tube and stored at 4°C.

[0076] Then, 100 mL of DPY liquid medium was prepared in a 500 mL Erlenmeyer flask equipped with a baffle, and 10 5 The bacteria were inoculated and cultured at 30°C and 150 rpm for 3 days.

[0077] The cells were then harvested using Miracloth, the medium was thoroughly squeezed, and the cells were then wrapped in aluminum foil and frozen completely at -80° C. The cells thus obtained were freeze-dried for more than 20 hours.

[0078] The freeze-dried cells were crushed with a pestle and then completely powdered using a Multi-Beads Shocker (registered trademark, Yasui Kikai Co., Ltd., 2000 rpm, 10 seconds).

[0079] 50 mg of the powdered dried cells was placed in a 50 mL disposable container, and then 0.50 mL of chloroform and 1.0 mL of methanol were added and thoroughly suspended by vibration stirring.

[0080] Next, 0.50 mL of chloroform was added again to the above disposable container, and the mixture was thoroughly suspended by vibration stirring, followed by ultrasonic treatment for 10 minutes.

[0081] Thereafter, 2 mL of 0.8 M (mol / L) KOH in methanol was added, and the mixture was incubated in a thermostatic chamber set at 42° C. for 30 minutes.

[0082] Next, 5 mL of chloroform and 2.25 mL of distilled water were added, and the mixture was thoroughly suspended by vibrating.

[0083] Finally, the mixture was incubated at 37° C. for 16 to 18 hours using a disposable stand to extract glycolipids.

[0084] [TLC] First, 4 mL of the lower layer of chloroform in the three-layer liquid containing the glycolipids extracted as described above was collected in a 15 mL disposable container. Next, the solvent was evaporated from the recovered chloroform phase, and the remaining solute was dissolved in 200 μL of an organic solvent prepared by mixing chloroform and methanol in a 2:1 ratio.

[0085] Next, a developing solvent was prepared by mixing chloroform containing 0.5% boric acid, methanol, and distilled water in a ratio of 65:20:2, and added to the developing tank (CAMAG). The developing solvent was adjusted so that the depth from the bottom of the developing tank was 0.5 cm.

[0086] Next, in order to uniformly fill the developing tank with vapor of the developing solvent, a filter paper of an appropriate size was placed upright in the developing tank, and the tank was left to stand with the lid on for 30 minutes or more.

[0087] Thereafter, 15 μL of the solution in which the above solute was dissolved was dropped onto a Silica gel 60 TLC plate (Merck) at a position 1 cm from the bottom in three separate portions.

[0088] The solvent was allowed to spread until the height reached 9.5 cm, and then the mixture was dried using a dryer.

[0089] Thereafter, the plate was immersed in methanol:sulfuric acid (=9:1) containing 0.2% orcinol, and then dried with a dryer.

[0090] Finally, the TLC plate was baked on a hot plate at 150°C.

[0091] FIG. 4 shows chromatographs of glycolipids obtained from various strains of Aspergillus oryzae.

[0092] As shown in FIG. 4, only the band representing glucosylceramide was observed in the wild-type strain into which the gceA gene had not been introduced and the control strain.

[0093] In contrast, in the system into which the Ptef or PpgkA primer and the gceA gene were introduced, no band indicating the presence of glucosylceramide was observed, but a band indicating the presence of galactosylceramide was observed.

[0094] From the above results, it was revealed that in the strain in which the galactosylceramide synthase shown in SEQ ID NO:1 was overexpressed, almost all of glucosylceramide was converted to galactosylceramide.

[0095] [Endogalactosylceramidase (EGALC) treatment] First, the organic solvent containing the glycolipids extracted by the above-mentioned treatment was subjected to a refrigerated centrifugal concentrator SpeedVac (registered trademark, manufactured by Thermo Fisher Scientific) to remove the organic solvent.

[0096] Next, 10 μL of 1% Triton (registered trademark) X-100 was added and sonicated using an ultrasonic disintegrator to dissolve the mixture. Note that the glycolipid was not completely dissolved.

[0097] Then, 4 μL of the solution was mixed with 2 μL of 0.5 M acetate buffer (pH 5.5, final concentration 50 mM), 4 μL of 0.15 mU endogalactosylceramidase (galactosidase), and 10 μL of water to make a total of 20 μL of solution, and the enzymatic reaction was carried out at 37° C. for 72 hours.

[0098] The reaction solution was then removed using a refrigerated centrifugal concentrator SpeedVac, 15 μL of methanol was added, and the mixture was sonicated using an ultrasonic disintegrator to dissolve the residue.

[0099] The entire amount of the solution was dropped onto a TLC plate, and the plate was developed with a developing solvent consisting of a 2:3:1 mixture of chloroform, methanol, and 0.02% CaCl2, and finally orcinol-sulfuric acid was sprayed onto the plate.

[0100] FIG. 5 is a chromatogram showing the result of reacting endogalactosylceramidase with glycolipids obtained from Aspergillus oryzae.

[0101] In FIG. 5, "GlcCer" is a system in which commercially available glucosylceramide is reacted with endogalactosylceramidase, and "GalCer" is a system in which commercially available galactosylceramide is reacted with endogalactosylceramidase.

[0102] The "overexpression strain" is a system in which endogalactosylceramidase was reacted with galactosylceramide extracted by the above-mentioned treatment from Aspergillus oryzae into which a plasmid containing PpgkA as a promoter had been introduced.

[0103] In addition, "RIB936" is a strain of Aspergillus oryzae that originally synthesizes galactosylceramide in addition to glucosylceramide.

[0104] Furthermore, "WT" is a RIB40 strain that does not originally synthesize galactosylceramide, as described above.

[0105] In addition, in FIG. 5, "-" indicates a negative control system that was not reacted with endogalactosylceramidase, and "+" indicates a system that was reacted with endogalactosylceramidase.

[0106] As shown in FIG. 5, first, in the case of "GlcCer", a single band B1 or B2 indicating glucosylceramide was confirmed regardless of the presence or absence of the reaction with endogalactosylceramidase.

[0107] In the system where endogalactosylceramidase was not reacted with "GalCer," band B3 indicating the presence of galactosylceramide was confirmed, whereas in the system where endogalactosylceramidase was reacted, band B4 indicating the presence of galactose released from galactosylceramide was confirmed.

[0108] On the other hand, in a system in which endogalactosylceramidase was not reacted with glycolipids extracted from the overexpression strain, no band indicating the presence of galactose was observed, whereas in a system in which endogalactosylceramidase was reacted, band B5 indicating the presence of galactose was observed.

[0109] From these results, it was confirmed once again that galactosylceramide was synthesized in the strain in which galactosylceramide synthase (see SEQ ID NO: 1) was overexpressed.

[0110] Furthermore, in a system in which glycolipids extracted from the RIB936 strain, which originally synthesizes galactosylceramide in addition to glucosylceramide, were not reacted with endogalactosylceramidase, no band indicating the presence of galactose was observed.

[0111] In contrast, in a system in which glycolipids extracted from the RIB936 strain were reacted with endogalactosylceramidase, band B6, indicating the presence of galactose, was confirmed.

[0112] Furthermore, in the RIB40 strain (WT), which does not naturally synthesize galactosylceramide (does not express galactosylceramide synthase), no band indicating the presence of galactose was observed, regardless of whether or not endogalactosylceramidase reaction occurred.

[0113] [Confirmation of gceA expression by RTPCR] (RNA extraction) 100 mL of DPY liquid medium was prepared in a 500 mL Erlenmeyer flask with a baffle, and 10 conidia were collected from the transformant strain of Aspergillus oryzae into which the above two types of plasmids with different promoters were introduced. 5 The bacteria were inoculated and cultured at 30°C and 150 rpm for 3 days.

[0114] Thereafter, the cells were collected using Miracloth, and the medium was thoroughly squeezed. An appropriate amount of the cells was then placed in a screw-cap microtube (SARSTEDT) and completely frozen at -80°C.

[0115] A chilled metal cone was placed in the above microtube, and the beads were disrupted using a Multi-Beads Shocker (registered trademark, Yasui Kikai Co., Ltd., 2000 rpm, 10 seconds).

[0116] The subsequent procedures were carried out using the RNeasy (registered trademark) Plant Mini Kit (50) manufactured by QIAGEN, following the protocol attached to the kit.

[0117] (cDNA synthesis) cDNA synthesis was performed using qPCR RT Master Mix (Toyobo Co., Ltd.), and the composition of the reaction solution was in accordance with the attached protocol.

[0118] (RTPCR) RTPCR was carried out using KOD-Plus-Neo (manufactured by Toyobo Co., Ltd.) The composition of the reaction solution was adjusted to 100 ng / μL by adding distilled water to the synthesized cDNA according to the attached protocol.

[0119] The primers for RTPCR used in this example are shown in Table 2.

[0120] [Table 2]

[0121] As a result of RTPCR, expression of the gceA gene was confirmed in the transformed strains of Aspergillus oryzae into which the above two types of plasmids with different promoters were introduced. [Industrial Applicability]

[0122] According to the present invention, there are provided a method for producing galactosylceramide using an enzyme derived from a filamentous fungus, and a filamentous fungus capable of mass-producing galactosylceramide. [Explanation of symbols]

[0123] S1: transformation step, S2: synthesis step, S3: extraction step, B1-B7: band

Claims

1. A method for producing galactosylceramide, comprising the step of contacting glucosylceramide with any one of the following enzymes (a) to (c) to convert the glucosylceramide into galactosylceramide: (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11; (b) an enzyme comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 11 and having an activity of converting glucosylceramide to the galactosylceramide; (c) an enzyme having an amino acid sequence having 60% or more sequence identity with any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having the activity of converting glucosylceramide to galactosylceramide;

2. The method for producing galactosylceramide according to claim 1, wherein the step of converting glucosylceramide into galactosylceramide is carried out within the cells of a filamentous fungus expressing any one of the enzymes (a) to (c).

3. The method for producing galactosylceramide according to claim 2 , further comprising a step of irradiating the filamentous fungus with ultraviolet light.

4. The method for producing galactosylceramide described in claim 2, further comprising a step of transforming the filamentous fungus so as to overexpress any one of the enzymes (a) to (c) prior to the step of converting glucosylceramide to galactosylceramide.

5. A filamentous fungus transformed to overexpress any one of the following enzymes (a) to (c): (a) an enzyme comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 11; (b) an enzyme comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having the activity of converting glucosylceramide to galactosylceramide; (c) an enzyme having an amino acid sequence having 60% or more sequence identity with any of the amino acid sequences shown in SEQ ID NOs: 1 to 11 and having the activity of converting glucosylceramide to galactosylceramide;

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